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| United States Patent |
5,149,425
|
|
Mazid
|
September 22, 1992
|
Affinity supports for hemoperfusion
Abstract
A method of coating chromatographic particulate supports to provide a
biocompatible outer layer of synthetic membrane-type film which prevents
the release of fines but permits the adsorption of components to an
affinity ligand is described. The matrix is provided with a membrane-type
coating, which prevents leaching of fines, with a pore size of at least 20
angstroms. The coating is applied to a solid particulate under conditions
where an integral membrane coat will be formed. It may also be necessary
to control the size and number of pores of the membranes by treating a
suspension of the solid support in a solvent which contains 0.1-1% of the
support weight of a biocompatible polymer along with 0.5-5% of the weight
of said polymer of a dissolved compatible pore-controlling component. The
solvent is then removed from the suspension and the membrane-coated
material used in extracorporeal treatment of body fluids or in other
chromatographic techniques. The coating process can be conducted before or
after the particulate support is functionalized and/or derivatized.
| Inventors:
|
Mazid; M. Abdul (Edmonton, CA)
|
| Assignee:
|
Chembiomed, Ltd. (Edmonton, CA)
|
| Appl. No.:
|
679801 |
| Filed:
|
April 3, 1991 |
| Current U.S. Class: |
210/198.2; 210/502.1; 210/635; 210/656; 502/402; 502/404 |
| Intern'l Class: |
B01D 015/08 |
| Field of Search: |
502/402,404
210/635,656,198.2,502.1,506,500.34,500.41
264/48,49
427/221,222,245,294,296
604/6
|
References Cited
U.S. Patent Documents
| 3577226 | May., 1972 | Elbert | 29/182.
|
| 3725113 | Apr., 1973 | Chang | 424/36.
|
| 3795313 | Mar., 1974 | Kirkland et al. | 210/198.
|
| 3808125 | Apr., 1974 | Good | 210/656.
|
| 3878092 | Apr., 1975 | Fuller | 210/656.
|
| 3953360 | Apr., 1976 | Morishita et al. | 502/402.
|
| 3954678 | May., 1976 | Maquisee | 502/402.
|
| 3983053 | Sep., 1976 | Courtney et al. | 502/402.
|
| 3983299 | Sep., 1976 | Regnier | 428/405.
|
| 4029583 | Jun., 1977 | Ho Chang et al. | 55/67.
|
| 4051300 | Sep., 1977 | Klein et al. | 210/500.
|
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|
| 4076892 | Feb., 1978 | Fennimore et al. | 502/402.
|
| 4104209 | Aug., 1978 | Mikes et al. | 521/62.
|
| 4137401 | Jan., 1979 | Lemieux et al. | 536/116.
|
| 4140652 | Feb., 1979 | Korshak et al. | 252/426.
|
| 4140653 | Feb., 1979 | Imura et al. | 502/402.
|
| 4163725 | Aug., 1979 | Sano et al. | 210/490.
|
| 4171283 | Oct., 1979 | Nakashima | 502/402.
|
| 4177038 | Dec., 1979 | Biebricher et al. | 210/656.
|
| 4238473 | Dec., 1980 | Lemieux et al. | 536/116.
|
| 4248736 | Feb., 1981 | Fuchigami et al. | 502/402.
|
| 4250256 | Feb., 1981 | Wielinger et al. | 435/810.
|
| 4332694 | Jun., 1982 | Kalal et al. | 252/189.
|
| 4352884 | Oct., 1982 | Nakashima | 435/180.
|
| 4362620 | Dec., 1982 | Lemieux et al. | 536/116.
|
| 4486376 | Dec., 1984 | Makino et al. | 210/500.
|
| 4557925 | Dec., 1985 | Lindahl et al. | 424/482.
|
| 4627992 | Dec., 1986 | Badenhop et al. | 264/49.
|
| 4629619 | Dec., 1986 | Lindahl et al. | 424/482.
|
| 4629620 | Dec., 1986 | Lindahl et al. | 424/482.
|
| 4634604 | Jan., 1987 | Tlustakova et al. | 210/198.
|
| 4663163 | May., 1987 | Hou et al. | 210/635.
|
| 4673734 | Jun., 1987 | Tayo et al. | 530/364.
|
| 4681870 | Jul., 1987 | Balint, Jr. et al. | 502/403.
|
| 4724207 | Feb., 1988 | Hou et al. | 435/180.
|
| 4761232 | Aug., 1988 | Bright | 210/500.
|
| 4772391 | Sep., 1988 | Baker et al. | 210/490.
|
| 4781838 | Nov., 1988 | Crassous et al. | 502/402.
|
| 4824678 | Apr., 1989 | Lindahl et al. | 424/473.
|
| Foreign Patent Documents |
| 0105579 | Apr., 1984 | EP | 210/198.
|
| 0129905 | Jan., 1985 | EP | 210/198.
|
| 0172579 | Feb., 1986 | EP | 210/198.
|
| 0165912 | Dec., 1987 | EP | 210/198.
|
| 3617672 | Dec., 1987 | DE | 210/198.
|
| 367063 | Feb., 1988 | DE | 210/198.
|
| 2239282 | Feb., 1975 | FR | 210/198.
|
| 2436607 | Apr., 1980 | FR | 210/198.
|
Other References
Refojo, J. Ap. Poly. Sci. (1965) 9:3417.intg.3125.
White, J. Phys. Chem. (1960) 64:1563-1565.
Schenkein et al., J. Clin. Invest. (1971) 50:1864-1868.
Lyle et al., J. Immunol. (1974) 113:517-521.
Terman et al., Clin. Exp. Immunol. (1977) 28:180-188.
Terman et al., New Eng. J. Med. (1981) 305:1195-1200.
Besa et al., Amer. J. Med. (1981) 71:1035-1040.
Messaikeh et al., Biological and Biomechanical Performance of Biomaterials
(1986) Christel et al., Eds., Elsevier, Amsterdam pp. 321-326.
Margel et al., Ap. Biochem. Biotechnol. (1986) 12:37-66.
Bensinger et al., Transfusion (1981) 21:335-342.
Bensinger et al., New Eng. J. Med. (1981) 304:160-162.
Bensinger et al., J. Clin. Apheresis (1982) 1:2-5.
Bensinger et al., Vox Sang (1985) 48:357-361.
Chang, Trans. Amer. Soc. Artif. Int. Organs (1980) 26:546-549.
Osterwalder et al., Blut (1986) 53:379-390.
Bussel et al., Plasma Ther. Trans. Technol. (1985) 6:461-464.
Raja et al., Trans. Amer. Soc. Artif. Internal Organs (1986) 32:102-103.
Bannett et al., Transplantation (1987) 43:909-910.
Hasirci et al., J. Biomed. Materials Res. (1986) 20:963-970.
Neumann et al., "Biocompatible Polymers, Metals, and Composites" (1983)
Szycher, editor; Technomic, PA pp. 53-80.
Thomas et al., "Biological and Biomechanical Performance of Biomaterials"
(1986) Christel et al., eds. Elsevier, Amsterdam pp. 379-383.
Zingg et al., Biomaterials (1981) 2:156-158.
Strong et al., Anal. Biomed. Eng. (1982) 10:71-82.
Boardman, J. Chromatog. (1959) 2:388-397.
|
Primary Examiner: Therkorn; Ernest G.
Attorney, Agent or Firm: Morrison & Foerster
Parent Case Text
This application is a division, of allowed application Ser. No. 07/270,950
filed, Nov. 9, 1988.
Claims
I claim:
1. An affinity matrix suitable for extracorporeal perfusion of a body fluid
for the selective removal of specific components or for chromatography
which comprises:
(a) a solid, particulate support derivatized to a specific affinity ligand,
wherein said derivatized support is
(b) coated with a biocompatible polymer,
wherein the polymer coating is an integral membrane-type coating, and
wherein the membrane coating has a pore size of at least 20 angstroms, and
wherein said coating prevents the leaching of fine particles from the
matrix.
2. The matrix of claim 1 wherein the support is an inert inorganic
particulate.
3. The matrix of claim 2 wherein the support is selected from synthetic,
mineral or biogenic silicates.
4. The matrix of claim 3 wherein the support is diatomite.
5. The matrix of claim 1 wherein the affinity ligand is a carbohydrate
moiety.
6. The matrix of claim 5 wherein the carbohydrate moiety is selected from
the oligosaccharide of FIG. 2.
7. The matrix of claim 6 wherein the carbohydrate moiety is A trisaccharide
(ATS).
8. The matrix of claim 7 wherein the affinity ligand is conjugated to the
support through a linker arm.
9. The matrix of claim 7 wherein the affinity ligand is conjugated to the
support through a carrier.
10. The matrix of claim 9 wherein the carrier is BSA.
11. The matrix of claim 1 wherein the polymer coating is selected from
polystyrene, polysulfone, polyether urethane, polydimethylsiloxane, PSMA
and BSA.
12. The matrix of claim 6 wherein the carbohydrate moiety is B
trisaccharide (BTS).
13. A coated support prepared by a method to coat a particulate support
with a biocompatible membrane-type coating of pore size of at least 20
angstroms, wherein the coating prevents the leaching of fine particles
from a matrix formed by the method, which method comprises:
preparing a suspension of the particulate support, wherein said support is
functionalized or derivatized with an affinity ligand,
in a solvent containing dissolved biocompatible polymer in an amount 0.1-1%
of the weight of the support; and
removing the solvent from the suspension, under conditions which result in
a thin, integral membrane.
14. A coated support prepared by a method to coat a particulate support
with a biocompatible membrane-type coating of pore size at least 20
angstroms, wherein the coating prevents the leaching of fine particles
from a matrix formed by the method, which method comprises:
preparing a suspension of the particulate support, wherein said support is
functionalized or derivatized with an affinity ligand,
in a solvent containing dissolved biocompatible polymer in an amount 0.1-1%
of the weight of the support, said solvent further containing a dissolved
compatible pore-controlling component in an amount 0.5-5% of the weight of
said polymer;
removing the solvent from the suspension; and
removing the pore-controlling component from the coating, under conditions
which results in a thin, integral membrane.
Description
TECHNICAL FIELD
The invention relates to the field of extracorporeal treatment of body
fluids and affinity chromatography. In particular, it concerns particulate
supports with protective membrane-type coatings suitable both for
hemoperfusion and for a range of less exacting chromatographic and sample
treatment techniques.
BACKGROUND ART
Convenient chromatographic supports which are stable, have high capacity,
and have low nonspecific adsorption, have long been sought. However, this
combination of properties is singularly difficult to achieve.
Single-substance supports such as charcoal or synthetic polymers are
nonspecific, and apparently cannot be made with both high capacity and
stability. Hybrid gels, such as that obtained by impregnating porous
silica with DEAE dextran, also suffer these defects.
A number of hybrid supports of this nature have been disclosed, for
example, in U.S. Pat. No. 4,673,734, directed to a mineral support
impregnated with aminated polysaccharide; U.S. Pat. No. 3,577,226,
describing polymerization and formation of a cross-linked polymer in the
pores of silica gel in situ; Boardman, N. K., J Chromatog (1959)
2:388-389, which describes the formation of a thin layer of resin in the
cavity of a porous support such as celite; and U.S. Pat. No. 3,878,092,
which also describes a polymeric-coated silica.
Uncoated forms of silica and porous glass, while providing a high porosity
and flow rate, are susceptible to degradation and nonspecific adsorption
of proteins because of silanol groups at the surface. In order to overcome
these disadvantages, hydrophilic polymeric coatings involving silane
coupling agents have also been disclosed. For example, U.S. Pat. Nos.
3,983,299 and 4,029,583 describe glycidoxypropyl trimethoxysilane attached
to a silica support. However, the adhesion qualities of the coating are
poor.
U.S. Pat. No. 4,332,694 describes the combination of a reactive epoxy with
an inorganic silica support. U.S. Pat. No. 4,352,884 describes coating of
inorganic materials with a copolymer of hydrophilic acrylate or
methacrylate along with a copolymerizable carboxylic acid or amine, and a
cross-linking agent, a procedure which resulted in insufficient binding to
the underlying substrate. U.S. Pat. No. 3,795,313 describes a siliceous
support coated with a methacryloxysilane; U.S. Pat. No. 3,808,125
describes a silica support chemically bonded to a copolymer made from a
coupling agent polymerized onto a polymeric backbone. In a different
approach, U.S. Pat. No. 4,070,348 describes copolymers of glycidyl and
amino-containing acrylates which are covalently modified with specific
ligands, such as enzymes or proteins.
European patent application No. 0172579, published Feb. 26, 1986 and the
U.S. Pat. No. 4,724,207, describe a modified silica support covalently
bonded to a synthetic copolymer which contains a polymer which can be
covalently coupled directly to the silica copolymerized with a material
which contains either an ionizable group, a hydrophobic material, or a
group capable of binding an affinity ligand. A related U.S. Pat. No.
4,663,163 describes and claims similarly modified polysaccharide supports.
Thus, these supports use a subsequently cross-linked polymeric coating as
a matrix to contain the specificity-conferring derivatization and as a
link to bind this material to the particulate organic or inorganic
support.
None of the foregoing-described chromatographic supports would be suitable
for use in hemoperfusion, either because the flow properties are
inadequate, because the supports are too unstable, or because nonspecific
binding is too prevalent. The foregoing approaches may also result in
hydrogels associated with the acrylic polymers which are inherently
disadvantageous as poorly adhering and unduly significant in modifying the
mechanical properties of the basic particles. For example, polyacrylic
hydrogels have calculated average pore radii of only 4-10 angstroms
(Refojo, M. F., J Ap Polym Sci (1965) 9:3417; White, M. L., J Phys Chem
(1960) 64:1563. Such pore sizes effectively exclude even small plasma
proteins such as albumin (158 .ANG..times.38 .ANG.) and gamma globulin
(235 .ANG..times.44 .ANG.).
For the foregoing reasons, the above-referenced chromatographic supports
are inappropriate for ex vivo treatment of biological fluids such as blood
or plasma. Suitability for such use requires high dimensional stability,
without any particulate release, high efficiency and capacity, and
biocompatibility, including lack of nonspecific adsorption. In presently
practiced techniques, nonspecific adsorbents, such as activated charcoal,
ion exchangers or resins, have been used for plasma perfusion, which is
easier to conduct than hemoperfusion but requires additional equipment to
separate cells from plasma and may also involve filtration of treated
plasma. Attempts to perform specific removal of blood components have been
reported, such as the passage of blood through a tube coated with a
specific immunoligand (Schenkein et al, J Clin Invest (1971) 50:1964; Lyle
et al, J Immunol (1974) 113:517). Terman et al, Clin Exp Immunol (1977)
28:180, describes an encapsulated sorbent coupled to nylon and used as
column, Terman et al, New Eng J Med (1981) 305:1195-1200, describes the
use of protein A bound to charcoal-collodion to treat solid tumors, and
Besa et al, Am J Med (1981) 71:1035, describes a stabilized protein A to
remove serum IgG in an autoimmune therapy. U.S. Pat. No. 4,681,870
discloses the use of a protein A silica immunosorbent to remove IgG from
biological fluids, a process which suffers from the disadvantage of the
release of "fines" during the ex vivo treatment. Messaikeh et al,
"Biological and Biomechanical Performance of Biomaterials" (1986),
Christel et al, eds, Elsevier, Amsterdam, pp. 321-326, describes use of
derivatives of polystyrene to remove Factor VIII:C ex vivo. Margel et al,
Ap Biochem Biotechnol (1986) 12:37-66, describes the use of derivatized
cross-linked agarose polyacrolein microspheric beads ("agarose acrobeads")
for specific hemoperfusion.
The use of specific affinity ligands coupled directly to an inorganic
support as a matrix for selective removal of materials from the plasma or
blood was described by Bensinger et al in a series of articles appearing
in Transfusion (1981) 21:335-342; New Eng J Med (1981) 304:160-162; J Clin
Apheresis (1982) 1:2-5; and Vox Sanq (1985) 48:357-361. In the latest of
these disclosures, the immunoadsorbent was thinly coated with collodion
applied by the method described by Chang, Trans Am Soc Artif Int Organs
(1980) 26:546-549 and the related U.S. Pat. No. 3,725,113, but the thin
collodion coating did not prevent the release of fines. Hydrophilic
coatings of nonspecific supports are described. Others have used similar
columns for removal of antibodies from human plasma (Osterwalder et al,
Blut (1986) 53:379-390; Bussel et al, Plasma Ther Transfus Technol (1985)
6:461-464) and from whole blood (Raja et al, ibid (1986) 22:102-103, and
Bannett et al, Transplantation (1987) 43:909-910). Attempts to coat
adsorbents also include the use of glow discharge to polymerize
hexamethyldisiloxane on the surface of activated charcoal granule for
hemoperfusion (Hasirci and Akovali, J Biomed Mater Res (1986) 20:963-970),
again failing to prevent the release of fines.
An additional problem is non-specific adsorption to the support. The
adhesion of various materials to polymeric substances has been studied. A
review of biocompatibility of various polymers is found in Neumann et al,
in "Biocompatible Polymers, Metals and Composites" (1983) (Szycher, ed),
Technomic, PA. For example, polystyrene has been shown to be a poor
adherent for cells (Thomas et al, in "Biological and Biomechanical
Performance of Biomaterials" (supra)). Platelet adhesion does not seem to
depend on surface smoothness or roughness (Zingg et al, Biomaterials
(1981) 2:156-158); however, for hydrophobic surfaces surface roughness
does affect cell adhesion under flow conditions (Strong et al, Anal Biomed
Engg (1982) 10:71-82).
The present invention provides a process for providing a controlled pore
coating with membrane-type physical properties conferring integrity and
mechanical strength, which is biocompatible for use in protecting affinity
supports to prevent the release of fines. The coating is thus consistent
with suitable mechanical properties of membranes, and is of appropriate
porosity to accommodate the penetration of blood proteins such as
antibodies or other materials for which an affinity ligand attached to the
support is reactive.
DISCLOSURE OF THE INVENTION
A hemoperfusion device containing a novel affinity adsorbent is provided
for the selective removal of specific substances from blood. The device
overcomes the limitations of prior art chromatographic supports intended
for extracorporeal immunoadsorption in clinical applications. The supports
are also useful for large-scale separation and purification of biological
macromolecules, based on affinity chromatographic techniques.
The specific removal of unwanted substances from the blood circulation by
extracorporeal hemoperfusion has profound medical significance as it is
far more desirable and convenient than plasmapheresis. When appropriate
affinity ligands are employed, only substances with specificity for
binding to the ligand are removed. The invention provides a support over
which the whole blood (or plasma) can be safely circulated and then
returned directly into the human body. The supports are thus
"biocompatible", i.e., these supports do not adversely affect any blood
components other than that specifically targeted. In addition, the
supports resist cellular and platelet adhesion, and prevent the release of
fines, which could otherwise lead to harmful embolism.
The adsorbent matrix of the invention is comprised of silica or other inert
particles to which an affinity ligand is attached, wherein the resulting
derivatized support is modified by a membrane-type coating. The adsorbent
matrix has properties suitable for selective removal of antibodies or
other undesirable substances directly from bloodstream or plasma by
extracorporeal immunoadsorption. The coating involves formation of
ultrathin, porous film of the synthetic membrane-type, obtained by a
technique similar to the phase inversion method commonly employed in
formation of membranes per se.
Because they more easily produce integral membranes, hydrophobic polymers
to obtain the membrane-type coating are preferred. In such instances, it
may be necessary to include, along with the polymer, a dissolved
compatible pore-controlling component in an amount 0.5-5% of the weight of
said polymer. The solvent is then removed to form the desired
biocompatible membrane-type coating, which coating has a pore size equal
to or greater than 20 angstroms. More hydrophilic polymer coatings may not
require the pore-controlling component. When coated with a membrane-type
coating, the support shows stability with respect to fines, at least after
further washing as needed to remove preliminary fine components. The
invention is also directed to the polymeric membrane-coated matrix either
underivatized, or derivatized to an affinity ligand.
Thus, in one aspect, the invention is directed to a method to coat
specific-affinity adsorbents which method comprises preparing a suspension
of a particulate solid inert support (which is underivatized or
derivatized with the affinity ligand) wherein the suspension is prepared
in a solvent containing dissolved polymer in an amount of 0.1-1% of the
weight of matrix, under conditions wherein, and followed by steps whereby,
the support is provided with a membrane-type coating to the polymer, said
membrane-type coating having the physical and mechanical properties of an
integral membrane, but wherein the membrane has pores of sufficient size
to permit access of the substances to be adsorbed to the affinity ligand.
Accordingly, the pore size should be at least 20 angstroms.
In another aspect, the invention relates to a device containing the coated
matrix of the invention and to methods of conducting extracorporeal
perfusion techniques with body fluids employing it. In particular, the
invention is directed to a method of hemoperfusion using the coated
supports of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an illustrative cartridge arrangement for use in hemoperfusion
using the immunoadsorbents of the invention.
FIG. 2 shows several affinity ligands useful in the invention method.
FIGS. 3 and 4 show shedding of fines from immunoadsorbents prepared using
coating protocols with varying amounts of pore-controlling component.
FIGS. 5A and 5B show the effect of conditions on fines shed from
polystyrene coated supports.
MODES OF CARRYING OUT THE INVENTION
A novel immunoadsorbent material is provided for, for example, the
selective removal of specific substances such as antibodies from the
bloodstream. Blood is withdrawn from a patient, circulated as whole blood
through the membrane-coated support of the invention to remove the
unwanted substance and the treated blood returned directly to the patient.
Alternatively, the blood cells may be separated from the whole blood
before treatment; the separated plasma is then treated by passing it over
the membrane-coated support and returning it to the patient. The separated
blood cells can also be reinfused into the patient directly or after
mixing with the treated plasma.
The membrane-coated support of the invention, when in use, generally
contains an affinity ligand. The ligand may be chosen from, for example,
chemically synthesized structures such as oligosaccharide determinants for
human blood groups, and can be covalently attached, directly or using a
linker, or non-covalently adsorbed preferably using a suitable carrier
molecule to a supporting particulate such as silica particles, porous
glass, etc. The immunoadsorbent thus obtained is modified by the membrane
type coating technique of the invention to impart properties more suitable
for whole blood hemoperfusion (or for use generally), by preventing fines,
but permitting the passage of whole blood containing the component to be
removed, and permitting the component to reach the specific ligand.
A. DEFINITIONS
As used herein, "biocompatible membrane-type coating" refers to a material
which has been coated to an inert support or to a functionalized or an
affinity derivatized support using the method of the invention and which
is, from the standpoint of composition, a synthetic or naturally derived
polymeric material which is chemically inert with respect to physiological
substances, and is biocompatible when used in contact with extracorporeal
fluids, including blood. It is believed that biocompatibility may be
enhanced by formation of a secondary albumin coating when in use under
certain conditions.
"Pore-controlling component" refers to a material which is soluble in both
the solvent used in the coating preparation, and the
gelation/wetting/leaching medium which follows the solvent evaporation
step described below. The pore-controlling component is also inert with
regard to forming a coating on the derivatized support. The
pore-controlling component is a nonsolvent or polymer-swelling agent whose
function is to control the size and/or number of pores, therefore
wettability and stability of the coated matrix. Examples include, for
example, low molecular weight polymers such as polyethylene glycol (PEG),
especially MW 300-20,000, and polypropylene glycol, polyvinyl alcohol,
polyvinyl pyrrolidone (PVP), or other low molecular weight, relatively
hydrophilic polymers. Also usable are nonionic detergents such as
Tween-20, Triton-X, various Solulans, electrolytes and the like.
"Inert support" refers to the particulate inert material to which the
coating polymer will be applied.
"Derivatized" support refers to the inert support which has conjugated
thereto an affinity ligand. The ligand may be directly bound covalently
(or noncovalently) to the support, or a linker and sometimes a carrier may
be employed.
"Functionalized" support refers to the inert support to which only a linker
moiety, or a moiety which provides a functional group for further
conjugation (e.g., a succinyl group to provide a carboxyl) is attached.
Functionalized support refers both to supports coated with the
membrane-type coating film of the invention, or not coated.
"Underivatized support" refers to support lacking added linkers or affinity
ligand. To fit the definition of "underivatized support" it also does not
matter whether the invention coating has or has not been applied.
"Linker" refers to a moiety which serves to space the affinity ligand from
the particulate support. The distinction between a "linker" and a general
moiety which provides a functional group for further conjugation is not
precise, nor is such precision important. Affinity ligands can be, and
often are, provided with linkers or "linking arms" before they are
conjugated to support; the linker will contain a functional group capable
of binding to a functional group either native to the support or provided
by an additional conjugating moiety.
B. MATERIALS
The invention provides a method to obtain a coated, protected affinity
adsorbent which is safe for use in hemoperfusion, as well as in the less
demanding procedures of plasmaperfusion and ordinary affinity
chromatography.
THE SUPPORT
The affinity matrix can be any derivatized particulate having a specific
ligand conjugated to or otherwise bound to the solid support particles.
For preparation of invention substrates, a wide variety of such solid
particulate supports has been described, as set forth in the Background
section above. The solid support particles useful in the invention are of
a variety of inert materials in particulate form, including various silica
derivatives such as silica powder, synthetic silicates such as porous
glass, biogenic silicates such as diatomaceous earth, silicatecontaining
minerals such as kaolinite, and so forth. Other suitable supports may
include synthetic resins or particulate polymers such as polystyrenes,
polypropylenes, polysaccharides, and so forth, including those useful as
coating polymers, as set forth below, or other commonly used
chromatographic supports such as alumina. Silaceous materials are
particularly preferred. Particularly preferred is calcined diatomaceous
earth of the cristobollite type, which contains surface hydroxy groups;
these are convenient for covalent attachment of ligands. Mesh sizes can
vary according to intended use from about 150 to 12; for hemoperfus n,
60/30 mesh is particularly preferred.
AFFINITY LIGANDS
The solid support (coated as described below, or uncoated) is directly
conjugated or otherwise covalently or noncovalently bound to an affinity
ligand--i.e., a material which has a specificity for a component of the
sample to be subjected to treatment with the affinity support. Such
ligands include immunoglobulins, fragments of immunoglobulins such as Fab,
F(ab').sub.2 or Fab' fragments, specifically interacting materials such as
biotin and avidin, bioreactive proteins such as protamine or enzymes such
as heparinase, nucleotide sequences, glycosaminoglycans such as heparin,
and carbohydrate moieties which represent antigenic or specifically
reacting domains of various biological materials. Some ligands, especially
the carbohydrate moieties, may be provided as conjugates, for example,
with proteins. In the specific adsorbents of the invention, this ligand is
coated passively or is conjugated to the solid support either directly, or
through a linker, before or after the polymeric coating is applied. The
linker is typically an organic bifunctional moiety of appropriate length
which serves simply to distance the ligand from the surface of the solid
support. Suitable ligands and linkers are those disclosed, for example, in
U.S. Pat. Nos. 4,137,401, 4,238,473 and 4,362,720.
The membrane-type coating which characterizes the affinity matrices of the
invention is an integral film which has the physical and mechanical
properties of a single membrane, but which contains pores of 20 angstroms
or more so that materials in the fluid to be treated will have ready
access to the affinity ligand attached to the support. The membrane itself
is formed from a polymer so that the film is integral over the particle
but leaves the required pores in the surface. A variety of polymers can be
employed, as set forth below, but the process followed and conditions used
to achieve the required pore size and the integral film will depend on the
nature of the polymer chosen.
MEMBRANE-TYPE COATINGS
Hydrophobic polymers are preferred, as integral membranes are more easily
obtained than with more hydrophilic materials. Because the final steps in
the coating and adsorption processes are conducted in aqueous media,
hydrophilic polymers tend to form coatings which "fall apart" unless the
membrane formation is very carefully controlled and managed. Thus, e.g.,
the prior art collodion coatings of Chang (supra) do not fall within the
scope of the invention, as they are applied without attention to
parameters that assure integral membrane formation nor do they use the
desired hydrophobic polymers.
If hydrophobic polymers are used, the formation of an integral
membrane-type coating is relatively facile--it is required only to use the
proper ratio of polymer to support and to evaporate the solvent carefully.
In order to assure formation of appropriate number and size of pores, it
may be required, when hydrophobic polymers are used, to include a
"pore-controlling substance". This material is typically a low MW polymer
and must be soluble in the solvent used for coating and in the
gelation/wetting/leaching medium which effects, among other things,
preliminary removal of fines.
Exemplary of polymers which can be used as the biocompatible membrane-type
coating in the method of the invention include hydrophobic materials, such
as polystyrenes, polyetherurethanes, polysulfones, fluorinated or
chlorinated polymers such as polyvinyl chloride, polyethylenes and
polypropylenes, polycarbonates and polyesters. The hydrophobic polymers
also include other polyolefins, such as polybutadiene,
polydichlorobutadiene, polyisoprene, polychloroprene, polyvinylidene
halides, polyvinylidene carbonate, and polyfluorinated ethylenes.
Copolymers are also useful as coatings, such as styrene-butadiene
copolymer or copolymer of .alpha.-methylstyrene and dimethylsiloxane.
Other polymers useful in the invention are synthetic or natural rubbers
and polysiloxanes (silicone polymers) containing aliphatic or aromatic
moieties such as polydimethylsiloxane, polyphenylmethylsiloxane, and
polytrifluoropropylmethyl siloxane. Also useful are polyacrylonitriles or
acrylonitrile-containing copolymers, such as poly
.alpha.-chloroacrylonitrile copolymers; polyesters, including polylactams
and polyarylates; polyalkylacrylates and polyalkylmethacrylate; alkyd or
terpinoid resins; polysulfones, including aliphatic-containing
polysulfonates, polyalkylene polysulfates. More hydrophilic polymers
include polyalkylene glycols, such as polyethylene glycol and
polypropylene glycol, of relatively high molecular weight; polyarylene
oxides; nylons, polyvinyl alcohols, and polyphosphates, such as
polyethylene methylphosphate, and the like, can also be used. For all of
the above, copolymers, including block interpolymers, and grafts and
blends can be employed. Suitable biologically derived polymers include
polyhydroxy materials such as cellulosic polymers, proteins, such as serum
albumin and collagen, glycosaminoglycans, and the like.
The polymers, especially polysiloxanes, polyhydroxy materials, and
proteins, may be cross-linked either after the coating is applied or
during the in situ formation of the coating.
The performance of the matrix for adsorption of larger moieties reactive
with the affinity ligand and its wettability are enhanced by the inclusion
of pore-controlling components in some circumstances as further described
below.
If hydrophilic polymer coatings are used, it is inadvisable to add a
pore-controlling component since integrity of the membrane is already
difficult to achieve. For hydrophobic polymers, addition of a
pore-controlling component may not be needed if the interaction of the
ligand and substance to be adsorbed involves sufficiently small-sized
moieties. For example, using a polystyrene coating, pores of sufficient
size to permit IgM to be adsorbed to an affinity ligand conjugated to
support are formed without this additional component.
C. THE COATING PROCESS
The supports of the invention are characterized by having a membrane-type
coating which is integral over the surface of the membrane and which
contains pores of the correct size. The process to obtain this coating is
critical to provide the desired characteristics wherein the number and
size of the pores in the membrane-type coating film are controlled, and
the wettability and stability of the support are controlled also by the
membrane. In the process for preparing this membrane-type coating,
therefore, the composition of the coating solution, including the nature
and concentration of the polymer used and the conditions of evaporation
and gelation, including the composition and nature of the
gelation/leaching bath medium are critical to obtaining these results.
Methods to obtain integral membranes in general are known in the
membrane-forming art, and these methods are applied to the affinity
supports of the invention.
The derivatized support is coated with a membrane-type film of the
biocompatible polymer with pores of at least 20 .ANG. according to a
method appropriate for the choice of polymers. The coating procedure can
be conducted before or after functionalization or before or after
derivatization of the support to the specific ligand, since the pore size
of the membrane-type coating can be controlled in the method of the
invention, and can thus be adjusted to permit subsequent derivatization of
the support.
In the invention method, the protective coating membrane is thus applied to
the derivatized, functionalized, or underivatized support by supplying the
polymer coating in a solvent medium which contains an amount of polymer
appropriate to obtain the desired thickness of an integral film. This
amount is a weight of polymer which is 0.1-1% of the weight of particulate
support, for particles of 12-150 mesh. After thorough mixing and
incubation for a suitable period, typically 15-30 min, the solvent is
evaporated under vacuum until the polymer-coated particles are dry. As is
understood in the art, the polymer may be cross-linked (or not) during the
coating process, or immediately thereafter, preferably before evaporation
of solvent. The dried particles are then wetted in a
gelation/wetting/leaching medium, typically an aqueous medium, to effect
gelation of polymer, leaching of nonintegral components, and to remove
fines already present. By adjusting the parameters of polymer solution
composition, temperature, time of incubation, rate of solvent evaporation,
gelation conditions etc., to mimic those typically used in the formation
of thin porous membranes, the resulting film has physical integrity.
If the polymer is relatively hydrophilic, such as cellulose acetate, nylon,
serum albumins, and the like, pore formation of adequate size may occur
automatically. It is more difficult, however, to achieve an integral
coating. When hydrophobic polymers, such as polystyrene and polysulfone,
are used, the process may have to be modified in order for the coating to
have pores of 20 angstroms or more. This is achieved through the addition
of a pore-controlling component to the coating mixture. Similar to the
procedure above, when a pore-controlling component in effective
concentration is present, after incubation with agitation for a suitable
time period, the solvent is evaporated, and the dried solid is resuspended
in a gelation/wetting/leaching aqueous medium to remove fines, including
uncoated polymeric gels, pore-controlling nonsolvent component, and any
residual solvent.
Typically, the process is conducted by suspending the solid support in a
solution containing both polymer and pore-controlling component. The
concentration of polymer represents 0.1-1%, preferably 0.5%, of the weight
of solid support suspended; the pore-controlling component is present in
an amount of 0.5-5%, preferably about 1% of the weight of the added
biocompatible polymer; hence it is present at a substantially lower
concentration. The choice of pore-controlling component depends, of
course, upon the choice of polymer, the appropriate solvent for polymer,
as well as the gelation/wetting/leaching medium. The pore-controlling
component must be compatible with the polymer and must be soluble in both
solvent and with the gelation/wetting/leaching medium.
In a typical protocol, the polymeric material is dissolved at a
concentration of approximately 2 g/l of a suitable solvent with warming if
necessary, and the appropriate amount, typically around 20 mg (about
0.5-5% of the weight of the polymer which will form the membrane coat) of
the pore-controlling component is added while stirring. When both
components are dissolved, the dried solid support, approximately 400 g/l,
or 100-1000 times the weight of the polymer which will form the membrane
coat, is suspended by gentle agitation in the solution. The suspension is
then gently agitated for a suitable time, approximately 15-30 min, and the
polymer may then be cross-linked (or not) before evaporation of the
solvent under vacuum. Both agitation and evaporation are conveniently
conducted in a rotovap. The temperature may be increased during the last
stages of evaporation, and the coated matrix should appear completely dry.
The coated matrix is then cooled gradually to room temperature and
suspended in a gelation/wetting/leaching medium, typically a water bath
which may contain other components helpful in controlling pore size, such
as alcohols (e.g., ethanol), acids (e.g., sulfuric acid), and the like,
depending on the nature of polymer, for a suitable time, typically 1-2 hr,
preferably under ambient conditions or at temperatures of about
4.degree.-40.degree. C., depending on the polymer. The matrix, if properly
coated, should not show visible gelation or precipitation during this
wetting process.
Whether a pore-controlling component has been included or not, the
suspended matrix is then washed thoroughly with leaching medium such as
water in order to eliminate any fine particles or traces of the coating
mixture, including any pore-controlling component or swelling agent and
residual solvent.
The washed matrix is then dried by suction, and then by heating to about
60.degree. C. to constant weight.
If the matrix has not yet been derivatized to the specific ligand, the
derivatization process is then conducted, appropriately, on the suspended
membrane-coated matrix.
D. CHARACTERISTICS OF THE COATED MATRIX
The coated matrices of the invention are of suitable capacity, porosity,
handling characteristics, stability, and specificity, if desired, to be
useful, not only in standard chromatographic procedures, but also in
extracorporeal treatment of biological fluids. Accordingly, the materials
are biocompatible in that they are capable of effecting the required
separation or removal without disturbing any accompanying biological
components.
On a molecular level, the materials for use in affinity chromatography or
perfusion can be described as particulate solid supports to which are
adsorbed or covalently bound, either directly or through linking arms, an
appropriate affinity ligand. The entire derivatized support is coated with
a thin integral membrane-type film of polymeric material having a pore
size appropriate to the material for which binding is intended. Suitable
pore sizes are of the order of 20 angstroms or greater for removal of
specific substances such as proteins or antibodies from biological fluids.
The materials of the invention are characterized by the attachment of the
affinity ligands to the particulate support, rather than to a polymer
coating, and by the porous, thin, enveloping coat. (Also included in the
invention are comparably coated supports which have not been derivatized
to affinity ligand. These are intermediates in the formation of the
matrices of the invention or can be used in non-specific procedures.)
E. USE OF THE COATED SUPPORTS
In general, the affinity matrices of the invention are used in a manner
similar to affinity chromatographic supports for standard chromatographic
procedures, as well as for extracorporeal perfusion of biological fluids
such as plasma and blood. For this latter purpose, a preferred arrangement
utilizes a cartridge for packaging of the matrix typified by that shown in
FIG. 1. As shown, the cartridge consists of a cylindrical body, a pair of
end caps, a pair of mesh screens at either end, and protective cap plugs.
A variety of similar designs can be used, but the materials of the
cartridge components must themselves be biocompatible, such as
polyethylene, teflon or lexan polycarbonates. The loaded cartridge is
assembled by placing a mesh screen over one end of the cylinder and
fitting an end cap over the screen. The cylinder is then filled with the
affinity matrix of the invention, and assembly is completed by placing the
other mesh screen and fitting the other end cap in place. Generally, the
packing is done under sterile conditions or the cartridge together with
its contents can be sterilized after packing, for example, by ethylene
oxide sterilization. The affinity matrix is then wetted by pumping in
pyrogen-free normal saline and washing to insure the removal of any small
particles that have been dislodged from the matrix or left during
preparation.
The cartridge is then filled with a 1% solution of human serum albumin in
normal saline and stored at 4.degree. C. When ready for use, the cartridge
is washed with 0.9% sodium chloride solution to which has been added a
suitable anticoagulant, such as ACD-A containing heparin in an effective
amount. For a 250 ml cartridge, for example, this is approximately 1 l of
the sodium chloride solution to which 150 ml of ACD-A containing 6,000
units of heparin has been added.
In use, the subject's blood or plasma is passed through the cartridge by
placing a shunt, e.g., a loop of plastic medical grade tubing, in the
patient's arm and circulating the blood through the cartridge with a
direct return to the patient. Alternatively, the blood can be circulated
through the tubing by a continuous-flow blood separator such as an IBM
Model 2997 or a semipermeable membrane separator to allow passage of
plasma separated from the cellular components. The plasma is then passed
through the cartridge and returned directly, or after remixing with cells,
to the subject.
The coated haptenized supports can also be used for purification of desired
blood components or ligands from other biological fluids. The biological
fluid is tumbled with the appropriate specific coated adsorbent at a
temperature of 4.degree. C.-room temperature, and the adsorbed biological
material eluted using procedures appropriate to the particular ligand. For
elution of antibodies; for example, 2% ammonia in 0.15M saline or 0.2M
acetic acid adjusted to pH 2, are satisfactory. Other eluants which may be
employed are 1% ammonia in saline, 0.2M glycine hydrochloride, various
chaotropic ions, and 0.1M saccharide solution.
For removal of anti-A or anti-B antibodies, approximately 25 mg of the
matrix prepared as in Example 1 below is sufficient to remove all
antibodies from 1 ml of antisera of saline titer 1/64.
The matrix may be reused several times without loss of activity if
reequilibrated with starting buffer before application of the new sample.
For purification, as opposed to hemoperfusion, smaller particle size
(100/120 mesh) is preferred.
F. EXAMPLES
The following examples are intended to illustrate, but not to limit, the
invention. In general, the examples and associated preparation procedures
illustrate the attachment of the affinity ligand to the particulate
support and the coating of the derivatized, functionalized, or
underivatized support with the membrane-type coating of the invention. As
stated above, these two aspects of treatment of the solid support can be
conducted in any order. Suitable procedures for derivatizing glycoside
haptens to the particulate support through linking arms are described in
U.S. Pat. Nos. 4,137,401; 4,238,473; and 4,362,720, incorporated herein by
reference. Preparation A exemplifies the procedures employed in these
issued patents.
PREPARATION A: COUPLING OF TRISACCHARIDES TO AMINOSILATED DIATOMITE
A. Acid-washed diatomaceous earth was aminosilated using the procedure of
Westal and Filbert, Meth Enzymol (1974) 34B: 64 and Weetall, Nature (1969)
223: 959-960, as employed in U.S. Pat. Nos. 4,137,401 and 4,238,473, cited
above. The synthetic hapten, the 8-azidocarbonyloctyl derivative of the A
trisaccharide, IA of FIG. 2, (ATS), as described in the cited patent
4,362,720 (2.31 g) is dissolved in 30 ml dry DMF by stirring in a flask
placed in an acetone/dry ice bath under dry conditions. The temperature of
the bath is then adjusted to -25.degree. C. and 1.35 ml of 4.55M HCl in
1,4-dioxane is added, followed by 252 .mu.l of t-butyl nitrite. The
reaction mixture is stirred for 30 min and 1.2 ml of diisopropylethylamine
is added. The azido solution is then added to a suspension of the
aminosilated diatomite (3 kg) in 8.4 l of dry acetonitrile at -2.degree.
C. with rapid stirring for 30 min and continued slower stirring for 2.5
hr. The resulting haptenated diatomite is allowed to settle and the
solvent distilled off under vacuum. Haptenization of the support is
verified by the phenol-sulfuric acid method of DuBois et al, Anal Chem
(1956) 28: 350-356. Typically, a minimum value of 0.35 .mu.mol hapten/g
adsorbent is obtained.
Methanol (7.2 l) was added to the dry haptenized diatomite, stirred for 10
min under vacuum, and 0.18 l of acetic anhydride in 1 l methanol is added.
The stirring is continued for 1 hr and the reaction mixture left overnight
at room temperature. The solvent is removed by draining, and finally
distilled under vacuum. The resulting product is washed repeatedly until
the O.D. of supernatant, measured at 420 nm in a 1 cm path, is less than
0.1. Four liters saturated bicarbonate solution is added in the second
wash cycle to neutralize residual acetic acid. The washed product is dried
by vacuum filtration and again washed with 2 l methanol, spread onto
stainless steel trays, placed in an oven, and dried at 70.degree. C.
overnight. A typical yield is approximately 2.85 kg.
B. The procedure of paragraph A is similarly conducted using, in place of
the 8-azidocarbonyloctyl derivative of the A trisaccharide, the
corresponding B-trisaccharide, 1B of FIG. 2, or the oligosaccharides
numbered II-VII in FIG. 2.
EXAMPLE 1
Coating of Trisaccharide A Derivatized Support
A. Polystyrene Coated Derivatized Support Using PEG as Pore-Controlling
Component
Approximately 6.4 l of trichloroethylene is heated to 45.degree. C. and
14.25 g of polystyrene (m.w. 250,000) is added and dissolved. The
pore-controlling component, 0.1425 g of PEG-300 is added while stirring.
The haptenized support of preparation A is then added and the slurry
rotated on a rotovap evaporator for 20 min, followed by evaporation of the
solvent under vacuum. The temperature is increased to 60.degree. C. and
the evaporation of solvent continued until the matrix appears dry.
The matrix is gradually cooled to room temperature and suspended in water
for 2 hr under ambient conditions. The matrix is readily wetted, and
little or no visible gelation/precipitation occurs. The matrix is then
washed thoroughly with water until the supernatant is clear of any gelled
polymer and there is no visible indication of fines. The washed matrix is
suction-dried on Buchner funnels and dried in an oven at 60.degree. C. for
4 hr or more until constant weight is achieved. The yield of coated
product is typically 2.8 kg.
B. Alternate Coatings
Using the same general procedures as set forth in paragraph A of this
Example, but substituting other polymers for polystyrene, other or no
pore-controlling components for PEG, and an appropriate solvent, the
following coated matrices were prepared using 30/60 or 100/120 mesh
diatomite derivatized to ATS:
______________________________________
Pore-
Polymer Controlling
Designation
Coating Component Solvent
______________________________________
CF1-46B Cellulose None Acetone/
acetate (1%) water
CF1-47C2 Nylon-66 (1%) None Formic acid
CF1-47B2 PSMA (1%) None DMF
CF1-58A1 Polystyrene (1%)
None DMF
CF1-58B1 Polysulfone (1%)
None DMF
CF1-58C2 Polystyrene (1%)
PVP TCE
CF1-58D2 Polysulfone (1%)
PVP DMF
CF1-73A2 BSA (0.2%, cross-
None Phosphate
linked) buffer
CF1-73B2 BSA (0.5%, cross-
None Phosphate
linked) buffer
CF1-73C2 BSA (1.0%, cross-
None Phosphate
linked) buffer
CF2-19B Collodion (0.36%)
None EtOH
CF2-22A1 Polyether None DMF
urethane (0.5%)
CF2-27A PDMS (1%) None DCM
CF2-29 PVA (1%, cross-
None Water
linked)
______________________________________
EXAMPLE 2
ATS-Derivatized Diatomite Coated with Human Serum Albumin (HSA)
A solution of 0.40 g HSA (Fraction V, Sigma #A-1653) is prepared by
dissolving the protein slowly in a 1 L evaporation flask containing 200 mL
water. 80 g of ATS-derivatized diatomite (Preparation A) is added to this
solution and the flask is gently rotated for 1 hr. The supernatant is
decanted and the matrix is suction-dried on a Buchner funnel, a total of
78.2 mg of protein being recovered in the supernatant. (Alternatively, the
matrix may be dried completely under vacuum so that essentially all the
protein is deposited onto the matrix.)
The deposited or adsorbed protein is then cross-linked on the coated matrix
by adding 200 mL of 1.25% glutaraldehyde solution in 0.033M KH.sub.2
PO.sub.4. The mixture is stirred at a low speed for 30 mins and then left
overnight at room temperature. The settled matrix is decanted and rinsed
with 500 mL of water containing 1M NaCl to remove any protein bound only
loosely on the matrix. The cross-linking of proteins is evident from the
appearance of insoluble films around the flask. However, the amount of
protein-films as well as the proteins recovered by rinsing the matrix
after glutaraldehyde cross-linking is negligible (about 2 mg) so that the
amount of HSA immobilized onto the matrix is about 4 mg/g of matrix, or 5
mg/g when vacuum-dried initially.
The coated matrix is then washed extensively with water (3 L) to remove
residual glutaraldehyde and also to remove cloudiness due to fines until
visibly clear supernatant is obtained. Finally, the matrix is
suction-dried and let stand overnight at room temperature for air-drying
until constant weight is achieved. This product is then tested for fines
as well as for biological activity.
EXAMPLE 3
Coupling of Antigen ATS-BSA to Amino-silated Diatomite Precoated with
Polystyrene
Aminosilated diatomite, prepared as in Preparation A, is coated with
polystyrene, as described in Example 1. A 1 g sample of the resulting
coated, functionalized diatomite is suspended in 10 ml of 0.1M phosphate
buffer, pH 7, containing 2.5% glutaraldehyde and incubated for 1 hr at
10.degree. C. with end-over-end rotation, followed by washing extensively
with water to remove glutaraldehyde. A solution of 2.675 mg of ATS-BSA
conjugate (containing the equivalent of 0.54 .mu.m ATS) is prepared in the
same buffer, added to the glutaraldehyde-activated coated matrix, and
tumbled overnight at room temperature. The supernatant is decanted and the
matrix washed thoroughly with a solution of 1M NaCl in 0.1M phosphate
buffer, pH 7.
In one procedure, protein determination of the supernatant and wash showed
a loss of 0.43 .mu.mol ATS which was chemically coupled to 1 g of the
matrix via glutaraldehyde (BK7-20A). A similar preparation in which
ATS-BSA was coupled in the presence of glutaraldehyde gave 0.09 .mu.m
ATS/g (BK7-20A1). The conjugated matrix is washed with water and air-dried
at room temperature to constant weight.
EXAMPLE 4
Evaluation of Fines
Several procedures were used to determine the level of fine particles
removable from matrices of the invention by washing. In an initial rough
test, a 0.3 g sample is weighed and placed in a 5 ml test tube, followed
by addition of 3 ml of 0.1M PBS. The mixture is allowed to stand for 10
min and the tube then inverted 10 times. After 1 min settling, the
supernatant is transferred to a 1 cm cell to measure optical density at
420 nm.
The procedure is also modified to simulate conditions which might be
encountered during extracorporeal use of the affinity matrix.
Approximately 75 g of matrix is placed in the cartridge shown in FIG. 1,
and the cartridge is rotated end over end in a hematology mixer for 1 hr
prior to evaluation of fines. Care should be taken that the matrix
entirely fills the cartridge. The cartridge is then perfused with 0.9%
saline (filtered at 0.2 .mu.m for sterilization) at a flow rate of 40
ml/min using a Masterflex pump. A total of 10 l of saline is perfused
through the system and 20 ml samples are collected at every liter washing.
A particle count in particle/ml is made for 1) particles which are greater
than 5 .mu.m in diameter and 2) particles which are greater than 25 .mu.m
in diameter. Counts are determined using a Coulter Counter, Model ZM, with
C256 Channelizer. Control counts are performed on saline prior to
perfusion and prior to rotation of the cartridge.
FIG. 3 shows the results obtained for fines of diameter greater than 5
.mu.m with control samples--one of which is coated with an integral
membrane surrounding the particulate support (a positive control) and the
other, a matrix coated with 0.36% collodion, as described by Chang (supra)
(a negative control). Both controls are underivatized diatomite, and the
procedure of Example 1 is used for the positive control except that the
coating solution did not contain any pore-controlling component. As
expected, the polystyrene-coated matrix is relatively free of fines as
compared to the collodion-coated negative control.
FIG. 4 shows the results for underivatized polystyrene-coated diatomite
prepared as in Example 1, but with differing amounts of the
pore-controlling component, PEG-300. The results indicate that the
conditions of Example 1--i.e., 1% PEG-300 (the percentage being based on
total polymer coat applied)--give superior results to higher amounts of
the pore-controlling PEG-300 shown in FIG. 4 (4% and 28%).
FIG. 5A shows the results for three test compositions of FIG. 4 and the
polystyrene coated positive control of FIG. 3 with regard to particles
having diameters more than 25 .mu.m. All percentages of pore-controlling
component appeared to give similar counts of particles in this size range.
The procedure described above was further modified as set forth below to
simulate additional conditions which might be encountered in
extracorporeal treatment of fluid:
(a) Standard operating procedure with 75 g matrix contained in a cartridge,
rotated dry for 1 hr in a hematology mixer, and washed with 10 l saline at
40 ml/min. Samples collected at the beginning and after each liter wash.
(b) Samples collected after temporary interruption of flow for 15 min
subsequent to the above procedure (a).
(c) Samples collected after prolonged interruption of flow overnight,
subsequent to the procedure (b).
(d) Samples collected after additional physical disturbance of the
cartridge caused by simulated transportation, subsequent to the above
procedure (c).
(e) Samples collected after increased physical disturbance introduced by
rotation for 1 hr under wet conditions, subsequent to procedure (d).
(f) Samples collected after subjecting the cartridge containing the matrix
to an extreme condition of physical disturbance by sudden impacts under
simulated conditions, subsequent to procedure (e).
(g) Samples collected after storage of the matrix at -20.degree. C. for 72
hr, subsequent to the standard procedure with CF2-14A followed by air
drying.
(h) Samples collected after incubating the cartridge overnight at
37.degree. C., subsequent to (g) without any other treatment.
FIGS. 5A and 5B show the results, under certain of these specified
conditions, and as shown in both figures, for two matrices: CF2-14A, which
is 30-60 mesh diatomite with a 0.5% polystyrene coat modified with a 4%
PEG-300 pore-controlling component, and CF2-16, which is the same as
CF2-14A, except that only 1% PEG-300 was used. The additional treatments
did not appear to affect markedly the particle count for the larger
particles (FIG. 5A), but the smaller particle count could be, at least
initially, dramatically affected (FIG. 5B). This happens particularly if
the matrix is kept under wet conditions for a prolonged period of time (15
days or so), as was the case with sample CF2-14A tested under condition
(g).
EXAMPLE 5
Assay for Biocompatibility: Hemolysis and Adsorption of Essential Blood
Components
Platelets are thought to be particularly adherent when polymeric substrates
are contacted with blood. The extent of platelet adhesion to
polystyrene-coated diatomite and similar matrices haptenized with the A
trisaccharide has been determined. A 0.5 g portion of matrix was incubated
with 3.75 ml of blood or saline at 37.degree. C. by end-over-end rotation
for 30 min, and the supernatants were counted for platelets (PLT), white
blood cells (WBC), red blood cells (RBC) and hemoglobin (HGB). Saline was
used to determine background (.ltoreq.1.times.10.sup.9 /1); fresh blood
was used as control.
Test conditions assume that 100 g of matrix would be used over 4 hr for
extracorporeal treatment of 6 l of whole blood from an average adult;
end-over-end rotation approximates physical disturbance of flow through
the cartridge. The results are shown in Table 3.
TABLE 3
______________________________________
Effect of Polystyrene-Coated Matrices
on Various Components of Human Blood
Matrix or PTL .times.
WBC .times.
RBC .times.
HGB,
Sample Used 10.sup.9 /l
10.sup.9 /l
10.sup.12 /l
g/dl
______________________________________
1) Fresh 350 5.1 4.09 12.8
Blood
2) Polystyrene- 376 5.9 3.95 12.9
coated di-
atomite 30/60,
CF2-14B
3) Polystyrene- 353 4.8 3.3 10.5
coated diato-
mite 30/60,
haptenized
with A-tri-
saccharide
(CF2-20A)
4) (3) Above 391 5.3 3.95 12.9
pre-incubated
with 1% HSA
______________________________________
A control (4) used polystyrene-coated matrix haptenized with A
trisaccharide preincubated with 1% HSA in saline to obtain a secondary
coating. HSA has been shown to reduce platelet adhesion to polymer
surfaces (Neumann et al, supra). These results show there is little if any
nonspecific adsorption of essential blood components to the matrix.
The matrices were also tested to determine whether appreciable hemolysis
occurred when blood is placed in contact with them. Details of the
hemolysis assay are as follows:
One or two grams of matrix were placed separately in test tubes containing
5 or 10 mL of 0.9% saline. A 0.1 mL sample of human blood previously
collected was added directly to each tube containing 1 gm matrix and
saline or to 5 mL of saline extracted from the mixture of 2 g matrix
incubated at 70.degree. C. for 24 hr. Similarly, 0.1 mL blood was added to
a tube of saline which acted as the negative control (no hemolysis) and
also to a tube of distilled water which acted as the positive control
(100% hemolysis). The contents of all the tubes were gently mixed and
incubated in a water bath at 37.degree. C. for one hr. After removing the
samples of matrix (direct contact method), the blood-saline mixtures were
centrifuged at 2,200 rpm for 5 min, and the absorbance of each supernatant
solution was determined spectrophotometrically at 545 nm. Percentage
hemolysis was determined as the difference between the absorbances of the
test and negative controls divided by the absorbance of the positive
control, times 100. By the extraction method the results were in the range
of 0.13-0.32%; by the direct method 0.53-0.73% hemolysis were noted for
CF2-14B and CF2-20A. (Less than 5% is considered non-hemolytic).
EXAMPLE 6
Efficacy of Coated Matrix in Specific Adsorption
An in vitro hemagglutination assay was used to show efficacy of the coated
matrix in adsorbing an illustrative ligand antibody to ATS. 25 mg of
matrix, haptenized to A trisaccharide, is incubated with 0.5 ml O-plasma
serum (which contains anti-A.sub.1) with rotation on a hematology mixer
for 1 hr at room temperature (21.degree. C.). The supernatant is removed
and tested for hemagglutination by IgM and IgG in serial dilutions with A
antigen-bearing red blood cells.
The results are shown in Table 4.
TABLE 4
______________________________________
Adsorption of anti-A.sub.1 by Coated Matrices
Solvent/
Material Nonsolvent in
Human anti-A.sub.1 Titers
Tested Coating Solution
IgM IgG IgM IgG
______________________________________
O-plasma 1:128 1:512
1:128 1:512
BK7-17
0.5% Polystyrene-
TCE/PEG-300 64 256 128 256
coated diatomite
(30/60) BK7-20
(negative control)
Haptenized (not coated) 16 32 16 64
diatomite
(30/60) (0.7
.mu.mole ATS/g)
AM8-40IA
(positive control)
0.5% Polystyrene-
TCE/PEG-300 8 32 16 32
coated haptenized
diatomite (30/60)
CF2-19A
0.5% Polyether
DMF/none 32 128 32 128
urethane-coated
haptenized
diatomite
(30/60) CF2-22A1
0.36% Collodion-
EtOH/none 32 64 32 64
coated hapten-
ized diatomite
(30/60) CF2-19B
Precoated diato-
TCE/PEG-300 16 64 16 64
mite coupled with
ATS-BSA after
glutaraldehyde
activation (0.43
.mu.mole ATS/g)
BK7-20A
Precoated diato-
TCE/PEG-300 8 32 8 32
mite coupled with
ATS-BSA in
presence of
glutaraldehyde
(0.09 .mu.mole
ATS/g)
BK7-20A1
1% PDMS-coated
DCM/none 8 16
haptenized diato-
mite (30/60)
CF2-27A
1% PVA-coated
H.sub.2 O/cross- 8 16
haptenized diato-
linking
mite (30/60)
reagents
CF2-29
Haptenized diato-
(not coated) 2 4
mite (100/120
mesh) (0.59 .mu.mole
ATS/g)
AS7-30 (positive
control)
1% cellulose
Acetone/H.sub.2 O 2 2
acetate-coated
diatomite
CF1-46B
1% Nylon-66
HCOOH/none 4 2
coated diatomite
CF1-47C2
1% PSMA-coated
DMF/none 8 16
diatomite
CF1-47B2
1% Polystyrene-
DMF/none 2 4
coated diatomite
CF1-58A1
1% Polysulfone-
DMF/none 2 4
coated diatomite
CF1-58B1
1% Polystyrene-
TCE/PVP 2 4
coated diatomite
CF1-58C2
1% Polysulfone-
DMF/PVP 2 4
coated diatomite
CF1-58D2
0.2% BSA-coated
Phosphate buffer/ 4 2
diatomite cross-linking
CF1-73A2 reagent
0.5% BSA-coated
Phosphate buffer/ 8 4
diatomite cross-linking
CF1-73B2 reagent
1.0% BSA-coated
Phosphate buffer/ 32 16
diatomite cross-linking
CF1-73C2 reagent
______________________________________
Polystyrene-coated immunoadsorbent A is clearly as effective as its
uncoated haptenized form. Immunosorbents with reduced particle size
(100/120 mesh) also show increased effectiveness; the results also
indicate that the method of conjugation of antigen (ATS-BSA) affects
efficacy.
EXAMPLE 7
Performance in Simulated Hemoperfusion
To simulate extracorporeal perfusion procedures pooled porcine blood (6 l)
obtained from a local slaughterhouse was anticoagulated further with a
solution of heparin and sodium citrate in normal saline and continuously
recirculated through a cartridge containing 150 g of the polystyrene
coated A trisaccharide-derivatized diatomite, prepared in Example 1, at 40
ml/min at room temperature (21.degree. C.) for 4.5 hours. Samples were
collected at half-hour intervals, and analyzed for total protein, albumin,
bilirubin, cholesterol, alkaline phosphatase and lactate dehydrogenase.
Very little or no changes in concentration of these components was found.
Antibody titers in the perfused blood were determined using human A.sub.1
RBC as described in Example 6. Initial titers for anti-A.sub.1 (IgM/IgG)
in porcine plasma were 64/128; these values dropped to 16/32 after 2 hours
of hemoperfusion.
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